AIM: To investigate different methods of creating incomplete intestinal obstruction in a rat model and to compare their electrophysiologic, morphologic and histologic characteristics.
METHODS: Rat ileum was partially obstructed by the respective application of: braided silk (penetrated the mesentery and surrounded intestine); half ligation (penetrated directly and ligated 1/2 cross-section of the intestine); wide pipe (6 mm in width, surrounded the intestine); narrow pipe (2 mm in width, surrounded the intestine). A control was also included (no obstruction). Various behavioral and electrophysiologic variables, as well as morphologic and immunohistochemical observations were recorded by blinded investigators at different time points (12, 24, 48, 72 h), including daily general condition, ileal wet weight and circumference, macromorphous and micromorphous intestine, bowel movement capability in vivo and in vitro, slow wave and neural electrical activity, and the number of c-Kit positive interstitial cells of Cajal (ICC).
RESULTS: Despite being of a similar general condition, these methods resulted in different levels of obstruction in each group compared with the control at different time points (12, 24, 48, 72 h). However, these fields of the wide pipe rat showed significantly differences when compared with the other three obstructed groups at 12 to 72 h, including macroscopic and histological presentation, intestinal transit ratio and contractility, circumference and wet weight, amplitude and frequency of nerve electrical discharge and slow wave, and ICC numbers (all P < 0.01).
CONCLUSION: The wide pipe rat method is significantly more reliable and stable than the other methods of obstruction, demonstrating that use of the wide pipe method can be a useful model of incomplete intestinal obstruction.
Intestinal obstruction; Model; Comparative study; Electrophysiology; Morphology; Interstitial cells of Cajal
Posttranslational carbonylation of proteins by the covalent attachment of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is a biomarker of oxidative stress. Tandem mass spectrometry (MS/MS) has become an essential tool for characterization of this modification. Chemical tagging methods have been used to facilitate the immunoaffinity-based enrichment or even quantification of HNE-modified peptides and proteins. With MS/MS spectra of the untagged modified peptides considered as references, a comparative evaluation is presented focusing on the impact of affinity-tagging with four carbonyl-specific reagents (2,4-dinitrophenyl hydrazine, biotin hydrazide, biotinamidohexanoic acid hydrazide and N’-aminooxymethylcarbonylhydrazino D-biotin) on collision-induced dissociation of the tagged HNE-carbonylated peptides. Our study has shown that chemical labeling may not be carried out successfully for all the peptides and with all the reagents. The attachment of a tag usually cannot circumvent the occurrence of strong neutral losses observed with untagged species and, in addition, fragmentation of the introduced tag may also be introduced. Chemical tagging of certain peptides may, nevertheless, afford more sequence ions upon MS/MS than the untagged carbonylated peptide, especially when Michael addition of the lipid peroxidation product occurs on cysteine residues. Therefore, tagging may increase the confidence of identifications of HNE-modified peptides by database searches.
Protein carbonylation has been associated with various pathophysiological processes. A representative reactive carbonyl species (RCS), 4-hydroxy-2-nonenal (HNE), has been implicated specifically as a causative factor for the initiation and/or progression of various diseases. To date, however, little is known about the proteins and their modification sites susceptible to “carbonyl stress” by this RCS, especially in the liver. Using chemoprecipitation based on a solid phase hydrazine chemistry coupled with LC-MS/MS bottom-up approach and database searching, we identified several protein-HNE adducts in isolated rat liver mitochondria upon HNE exposure. The identification of selected major protein targets, such as the ATP synthase β-subunit, was further confirmed by immunoblotting and a gel-based approach in combination with LC–MS/MS. A network was also created based on the identified protein targets that showed that the main protein interactions were associated with cell death, tumor morphology and drug metabolism, implicating the toxic nature of HNE in the liver mitoproteome. The functional consequence of carbonylation was illustrated by its detrimental impact on the activity of ATP synthase, a representative major mitochondrial protein target for HNE modifications.
Latent HIV reservoirs are the primary hurdle to eradication of infection. Identification of agents, pathways and molecular mechanisms that activate latent provirus may, in the presence of highly active antiretroviral therapy, permit clearance of infected cells by the immune system. Promoter-proximal pausing of RNA polymerase (Pol) II is a major rate-limiting step in HIV gene expression. The viral Tat protein recruits human Super Elongation Complex (SEC) to paused Pol II to overcome this limitation. Here, we identify the bromodomain protein Brd4 and its inhibition of Tat-transactivation as a major impediment to latency reactivation. Brd4 competitively blocks the Tat–SEC interaction on HIV promoter. The BET bromodomain inhibitor JQ1 dissociates Brd4 from the HIV promoter to allow Tat recruitment of SEC to stimulate HIV elongation. JQ1 synergizes with another latency activator prostratin, which promotes Pol II loading onto the viral promoter. Because JQ1 activates viral latency without inducing global T cell activation, this and other closely related compounds and their antagonization of Brd4 to promote Tat–SEC interaction merit further investigations as effective agents/strategies for eliminating latent HIV.
Furanodiene (FUR) is a natural terpenoid isolated from Curcumae Rhizoma, a well-known Chinese medicinal herb that presents antiproliferation activities in several cancer cell lines. In this study, we demonstrated that FUR concentration dependently inhibits the cell proliferation of A549, NIH-H1299, and 95-D lung cancer cells. β-elemene, another terpenoid isolated from Curcumae Rhizoma, exhibited weaker antiproliferative effects in A549 and NIH-H1299 cells and activities similar to FUR in 95-D cells. FUR significantly inhibited colony formation in A549 and 95-D cells and upregulated both the mRNA and protein expression levels of binding immunoglobulin protein (BIP) and C/EBP homologous protein (CHOP), indicating that endoplasmic reticulum (ER) stress is induced. FUR treatment led to the accumulation of CHOP in the nucleus, which further confirms induction of ER stress. Furthermore, combined treatment of FUR with paclitaxel showed significant synergetic activities in NIH-H1299 and 95-D cells, suggesting its potential roles in combination therapy. These findings provide a basis for the further study of the anticancer effects in vivo and the internal mechanisms of FUR.
Chemokine binding to cognate receptors induces actin dynamics that are a major driving force for T cell migration and chemotactic motility. HIV-1 binding to the chemokine coreceptor CXCR4 initiates chemotactic signaling, mimicking chemokine-induced actin dynamics to facilitate infection processes such as entry, early DNA synthesis, and nuclear migration. Recently, we identified that HIV-triggered early actin polymerization is mediated through the Rac1-PAK1/2-LIMK1-cofilin pathway. Inhibition of LIMK1 (LIM domain kinase 1), a kinase phosphorylating cofilin, through shRNA knockdown decreases actin polymerization and T cell chemotaxis toward SDF-1. The LIMK1 knockdown T cells also supported lower viral entry, DNA synthesis and nuclear migration, suggesting a critical role of LIMK1-mediated actin dynamics in the initiation of HIV-1 infection. Surprisingly, LIMK1 knockdown in CEM-SS T cells did not lead to an overall change in the ratio of phospho-cofilin to total cofilin although there was a measurable decrease in the amount of actin filaments in cells. The decrease in filamentous actin in LIMK1 knockdown cells was found to mainly occur in polarized cap region rich in F-actin. These results suggest that LIMK1 may be involved in spontaneous actin polarization in transformed T cells. The inhibition of T cell chemotaxis by LIMK1 knockdown likely result from inhibition of localized LIMK1 activation and cofilin phosphorylation that are required for polarized actin polymerization for directional cell migration. The inhibition of HIV-1 infection by LIMK1 knockdown may also result from the decrease of actin-rich membrane protrusions that may be preferred viral entry sites in T cells.
LIMK1; cofilin; chemotaxis; SDF-1; CXCR4; HIV-1; CD4 T cells; Rac1; Pak1; Pak2
Anti-obesity drugs are widely used to prevent the complications of obesity, however, the effects of anti-obesity drugs on cardiovascular risk factors are unclear at the present time. We carried out a comprehensively systematic review and meta-analysis to assess the effects of anti-obesity drugs on cardiovascular risk factors.
Methodology and Principal Findings
We systematically searched Medline, EmBase, the Cochrane Central Register of Controlled Trials, reference lists of articles and proceedings of major meetings for relevant literatures. We included randomized placebo-controlled trials that reported the effects of anti-obesity drugs on cardiovascular risk factors compared to placebo. Overall, orlistat produced a reduction of 2.39 kg (95%CI-3.34 to −1.45) for weight, a reduction of 0.27 mmol/L (95%CI: −0.36 to −0.17) for total cholesterol, a reduction of 0.21 mmol/L (95%CI: −0.30 to −0.12) for LDL, a reduction of 0.12 mmol/L (95%CI: −0.20 to −0.04) for fasting glucose, 1.85 mmHg reduction (95%CI: −3.30 to −0.40) for SBP, and a reduction of 1.49 mmHg (95%CI: −2.39 to −0.58) for DBP. Sibutramine only showed effects on weight loss and triglycerides reduction with statistical significances. Rimonabant was associated with statistically significant effects on weight loss, SBP reduction and DBP reduction. No other significantly different effects were identified between anti-obesity therapy and placebo.
We identified that anti-obesity therapy was associated with a decrease of weight regardless of the type of the drug. Orlistat and rimonabant could lead to an improvement on cardiovascular risk factors. However, Sibutramine may have a direct effect on cardiovascular risk factors.
Rationale: The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known.
Objectives: To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis.
Methods: Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1f/f conditional knockout mouse after being exposed to silica or bleomycin.
Measurements and Main Results: TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β–induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1+/−) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1f/f mice reduced lung fibrosis.
Conclusions: YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB–dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.
nuclear factor-κB; α-smooth muscle actin; idiopathic pulmonary fibrosis
For an infecting viral pathogen, the actin cortex inside the host cell is the first line of intracellular components that it encounters. Viruses devise various strategies to actively engage or circumvent the actin structure. In this regard, the human immunodeficiency virus-1 (HIV-1) exemplifies command of cellular processes to take control of actin dynamics for the initiation of infection. It has becomes increasingly evident that cortical actin presents itself both as a barrier to viral intracellular migration and as a necessary cofactor that the virus must actively engage, particularly, in the infection of resting CD4 blood T cells, the primary targets of HIV-1. The coercion of this most fundamental cellular component permits infection by facilitating entry, reverse transcription, and nuclear migration, three essential processes for the establishment of viral infection and latency in blood T cells. It is the purpose of this review to examine, in detail, the manifestation of viral dependence on the actin cytoskeleton, and present a model of how HIV utilizes actin dynamics to initiate infection.
HIV-1; gp120; Nef; Actin; Cofilin; LIMK1; Arp2/3; CD4; CXCR4; Chemokine; Chemotaxis
Centrifugal inoculation, or spinoculation, is widely used in virology research to enhance viral infection. However, the mechanism remained obscure. Using HIV-1 infection of human T cells as a model, we demonstrate that spinoculation triggers dynamic actin and cofilin activity, probably resulting from cellular responses to centrifugal stress. This actin activity also leads to the upregulation of the HIV-1 receptor and coreceptor, CD4 and CXCR4, enhancing viral binding and entry. We also demonstrate that an actin inhibitor, jasplakinolide, diminishes spin-mediated enhancement. In addition, small interfering RNA (siRNA) knockdown of LIMK1, a cofilin kinase, decreases the enhancement. These results suggest that spin-mediated enhancement cannot be explained simply by a virus-concentrating effect; rather, it is coupled with spin-induced cytoskeletal dynamics that promote receptor mobilization, viral entry, and postentry processes. Our results highlight the importance of cofilin and a dynamic cytoskeleton for the initiation of viral infection. Our results also indicate that caution needs to be taken in data interpretation when cells are spinoculated; some of the spin-induced cellular permissiveness may be beyond the natural capacity of an infecting virus.
Resveratrol have been known to possess many pharmacological properties including antioxidant, cardioprotective and anticancer effects. Although current studies indicate that resveratrol produces neuroprotection against neurological disorders, the precise mechanisms for its beneficial effects are still not fully understood. We investigate the effect of anti-inflammatory and mechamisms of resveratrol by using lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells.
BV-2 cells were treated with resveratrol (25, 50, and 100 µM) and/or LPS (1 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10), Akt, mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) cascades, inhibitor κB-α (IκB-α) and cyclic AMP-responsive element-binding protein (CREB) were measured by western blot. Resveratrol significantly attenuated the LPS-induced expression of NO, PGE2, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nuclear factor-κB (NF-κB) in BV-2 cells. Resveratrol increased PTEN, Akt and mTOR phosphorylation in a dose-dependent manner or a time-dependent manner. Rapamycin (10 nM), a specific mTOR inhibitor, blocked the effects of resveratrol on LPS-induced microglial activation. In addition, mTOR inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of IκB-α, CREB, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK).
Conclusion and Implications
This study indicates that resveratrol inhibited LPS-induced proinflammatory enzymes and proinflammatory cytokines via down-regulation phosphorylation of NF-κB, CREB and MAPKs family in a mTOR-dependent manner. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of resveratrol.
Geoditin A, an isomalabaricane triterpene isolated from marine sponge Geodia japonica, has been demonstrated to induce apoptosis in leukemia HL60 cells and human colon HT29 cancer cells through an oxidative stress, a process also interfering with normal melanogenesis in pigment cells. Treatment of murine melanoma B16 cells with geoditin A decreased expression of melanogenic proteins and cell melanogenesis which was aggravated with adenylate cyclase inhibitor SQ22536, indicating melanogenic inhibition was mediated through a cAMP-dependent signaling pathway. Immunofluorescence microscopy and glycosylation studies revealed abnormal glycosylation patterns of melanogenic proteins (tyrosinase and tyrosinase-related protein 1), and a co-localization of tyrosinase with calnexin (CNX) and lysosome-associated membrane protein 1 (LAMP-1), implicating a post-translational modification in the ER and a degradation of tyrosinase in the lysosome. Taken together, potent anti-melanogenic property and the relatively low cytotoxicity of geoditin A have demonstrated its therapeutic potential as a skin lightening agent.
geoditin A; B16 melanoma cells; melanogenesis; tyrosinase
Appropriate diagnostics to monitor disease trends and assess the effectiveness and impact of interventions are essential for guiding treatment strategies at different thresholds of schistosomiasis transmission and for certifying elimination. Field validation of these assays is urgently needed before they can be adopted to support policy decisions of the national programme for control and elimination of schistosomiasis in P.R. China. We compared the efficacy and utility of different immunoassays in guiding control strategies and monitoring the endemic status of S. japonicum infections towards elimination.
A cross-sectional survey was conducted in seven villages with different transmission intensities settings to assess the performance and utility of three immunoassays, e.g., an indirect hemagglutination assay (IHA_JX), an enzyme linked immunosorbent assay (ELISA_SZ), and a dot immunogold filtration assay (DIGFA_SH). 6,248 individuals aged 6–65 years old who gave consent and supplied their stool and blood samples were included for data analysis. Results showed that ELISA_SZ performed significantly higher sensitivity (95.45%, 95%CI: 92.94–97.97%) than IHA_JX (87.59%, 95%CI: 83.51–91.49%) and DIGFA_SH (79.55%, 95%CI: 74.68–84.41%), especially in subgroups with very low infection intensity. The specificity of ELISA_SZ, IHA_JX, DIGFA_SH in 6–9 year olds with occasional exposure was nearly 90%. DIGFA_SH performed the highest screening efficacy for patients among three assays with overall positive predicative value of 13.07% (95%CI: 11.42–14.72%). We found a positive correlation of antibody positive rate of IHA_JX with results of stool examination in age strata (r = 0.70, P<0.001). Seropositivity of IHA_JX in children aged 6–9 years old showed an excellent correlation with prevalence of schistosome infection in the seven communities (r = 0.77, P<0.05).
Studies suggest that ELISA_SZ could be used to guide selective chemotherapy in moderate or low endemic regions. IHA_JX could be used to as a surveillance tool and for certifying elimination of schistosomiasis through monitoring children as a sentinel population.
Immunodiagnostic assays are widely applied in the field to control schistosomiasis in P.R. China as the prevalence and infection intensity of schistosome infections decrease. Field evaluations are urgently needed before they can be adopted to support policy decisions of the national programme for the control and elimination of schistosomiasis in P.R. China. We carried out a large scale cross-sectional survey in field settings with different transmission situations to validate immunodiagnostic tools that can be used to formulate new schistosomiasis elimination strategy in P.R. China. Regarding stool examination as gold reference, the validity and screening efficacy of each immunodiagnostic kit were calculated and compared with each other. The association of the prevalence of schistosomiasis and antibody positive rates determined by immunoassays were analyzed using Pearson's correlation coefficient values. The study indicates that which test to use with the elimination strategy is dependent on the purpose of testing, the endemic status of community and the resources available. And more sensitive methods need to be explored and used to target infected individuals for treatment or to eliminate schistosomiasis.
The mononuclear title molecule, [Sn(C7H6F)2(C3H3N2S3)2], has 2 symmetry. The SnIV atom, located on a twofold rotation axis, is in a skew trapezoidal–bipyramidal geometry, with the basal plane defined by two S,N-chelating 3-methylsulfanyl-1,2,4-thiadiazole-5-thiolate ligands. The apical positions are occupied by the C atoms of two 4-fluorobenzyl groups.
Ischemic postconditioning (IPO) has been demonstrated to attenuate ischemia/reperfusion (I/R) injury in the heart and brain, its roles to liver remain to be defined. The study was undertaken to determine if IPO would attenuate liver warm I/R injury and its protective mechanism.
Mice were divided into sham, I/R, IPO+I/R (occlusing the porta hepatis for 60 min, then treated for three cycles of 10 sec brief reperfusion consecutively, followed by a persistent reperfusion); L-NAME+ sham (L-NAME, 16 mg/kg, i.v., 5 min before repefusion); L-NAME+I/R; and L-NAME+ IPO. Blood flow of caudate and left lobe of the liver was blocked. Functional and morphologic changes of livers were evaluated. Contents of nitric oxide, eNOS and iNOS in serum were assayed. Concentration of eNOS, iNOS, malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in hepatic tissue were also measured. Expressions of Akt, p-Akt and HIF-1α protein were determined by western blot. Expressions of TNF-α and ICAM-1 were measured by immunohistochemistry and RT-PCR.
IPO attenuated the dramatically functional and morphological injuries. The levels of ALT was significantly reduced in IPO+I/R group (p < 0.05). Contents of nitric oxide and eNOS in serum were increased in the IPO+I/R group (p < 0.05). IPO also up-regulated the concentration of eNOS, activity of SOD in hepatic tissue (p < 0.05), while reduced the concentration of MDA (p < 0.05). Moreover, protein expressions of HIF-1α and p-Akt were markedly enhanced in IPO+I/R group. Protein and mRNA expression of TNF-α and ICAM-1 were markedly suppressed by IPO (p < 0.05). These protective effects of IPO could be abolished by L-NAME.
We found that IPO increased the content of NO and attenuated the overproduction of ROS and I/R-induced inflammation. Increased NO contents may contribute to increasing HIF-1α level, and HIF-1α and NO would simultaneously protect liver from I/R injury. These findings suggested IPO may have the therapeutic potential through Akt-eNOS-NO-HIF pathway for the better management of liver I/R injury.
The Childhood Adaptation Model to Chronic Illness: Diabetes Mellitus was developed to identify factors that influence childhood adaptation to type 1 diabetes (T1D). Since this model was proposed, considerable research has been completed. The purpose of this paper is to update the model on childhood adaptation to T1D using research conducted since the original model was proposed. The framework suggests that individual and family characteristics, such as age, socioeconomic status, and in children with T1D, treatment modality (pump vs. injections), psychosocial responses (depressive symptoms and anxiety), and individual and family responses (self-management, coping, self-efficacy, family functioning, social competence) influence the level of adaptation. Adaptation has both physiologic (metabolic control) and psychosocial (QOL) components. This revised model provides greater specificity to the factors that influence adaptation to chronic illness in children. Research and clinical implications are discussed.
Previous studies have observed fluorescently labeled HIV particles tracking along microtubule networks for nuclear localization. To provide direct evidence for the involvement of microtubules in early steps of HIV infection of human CD4 T cells, we used multiple microtubule modulators such as paclitaxel (originally called taxol; 1 μM), vinblastine (1 and 10 μM), colchicine (10 and 100 μM), and nocodazole (10 and 100 μM) to disturb microtubule networks in transformed and resting CD4 T cells. Although these drugs disrupted microtubule integrity, almost no inhibition of HIV-1 infection was observed. Our results do not appear to support an essential role for microtubules in the initiation of HIV infection of CD4 T cells.
The dipstick dye immunoassay (DDIA), recently commercially available in the People's Republic of China (P.R. China), is a rapid and simple test to detect human antibodies against Schistosoma Japonicum. Its performance and utility for screening schistosome infection in low endemic areas is little known. We therefore carried out a cross-sectional survey in seven villages with low endemicity of schistosomiasis in P.R. China and assessed the performance and utility of DDIA for diagnosis of schistosomiasis. Stool samples were collected and examined by the Kato-Katz method and the miracidium hatching technique. Serum samples, separated from whole blood of participants, were tested by DDIA.
6285 individuals aged 6-65 years old participated in this study, with a prevalence of schistosomiasis of 4.20%. Using stool examination as a gold reference standard, DDIA performed with a high overall sensitivity of 91.29% (95% CI: 87.89-94.69%) and also a high negative predictive value, with a mean value of 99.29% (95% CI: 98.99-99.58%). The specificity of DDIA was only moderate (53.08%, 95% CI: 51.82-54.34%). Multivariate analysis indicated that age, occupation and history of schistosome infection were significantly associated with the false positive results of DDIA.
DDIA is a sensitive, rapid, simple and portable diagnostic assay and can be used as a primary approach for screening schistosome infection in areas of low endemicity. However, more sensitive and specific confirmatory assays need to be developed and combined with DDIA for targeting chemotherapy accurately.
The Human Genome Project has concluded, but its successful completion has increased, rather than decreased, the need for high-throughput DNA sequencing technologies. The possibility of clinically screening a full genome for an individual's mutations offers tremendous benefits, both for pursuing personalized medicine as well as uncovering the genomic contributions to diseases. The Sanger sequencing method—although enormously productive for more than 30 years—requires an electrophoretic separation step that, unfortunately, remains a key technical obstacle for achieving economically acceptable full-genome results. Alternative sequencing approaches thus focus on innovations that can reduce costs.
The DNA sequencing by synthesis (SBS) approach has shown great promise as a new sequencing platform, with particular progress reported recently. The general fluorescent SBS approach involves (i) incorporation of nucleotide analogs bearing fluorescent reporters, (ii) identification of the incorporated nucleotide by its fluorescent emissions, and (iii) cleavage of the fluorophore, along with the reinitiation of the polymerase reaction for continuing sequence determination. In this Account, we review the construction of a DNA-immobilized chip and the development of novel nucleotide reporters for the SBS sequencing platform.
Click chemistry, with its high selectivity and coupling efficiency, was explored for surface immobilization of DNA. The first generation (G-1) modified nucleotides for SBS feature a small chemical moiety capping the 3′-OH and a fluorophore tethered to the base through a chemically cleavable linker; the design ensures that the nucleotide reporters are good substrates for the polymerase. The 3′-capping moiety and the fluorophore on the DNA extension products, generated by the incorporation of the G-1 modified nucleotides, are cleaved simultaneously to reinitiate the polymerase reaction. The sequence of a DNA template immobilized on a surface via click chemistry is unambiguously identified with this chip-SBS system.
The second generation (G-2) SBS system was developed based on the concept that the closer the structures of the added nucleotide and the primer are to their natural counterparts, the more faithfully the polymerase would incorporate the nucleotide. In this approach, the polymerase reaction is performed with the combination of 3′-capped nucleotide reversible terminators (NRTs) and cleavable fluorescent dideoxynucleotides (ddNTPs). By sacrificing a small amount of the primers permanently terminated by ddNTPs, the majority of the primers extended by the reversible terminators are reverted to the natural ones after each sequencing cycle. We have also developed the 3′-capped nucleotide reversible terminators to solve the problem of deciphering the homopolymeric regions of the template in conventional pyrosequencing. The 3′-capping moiety on the DNA extension product temporarily terminates the polymerase reaction, which allows only one nucleotide to be incorporated during each sequencing cycle. Thus, the number of nucleotides in the homopolymeric regions are unambiguously determined using the 3′-capped NRTs.
It has been established that millions of DNA templates can be immobilized on a chip surface through a variety of approaches. Therefore, the integration of these high-density DNA chips with the molecular-level SBS approaches described in this Account is expected to generate a high-throughput and accurate DNA sequencing system with wide applications in biological research and health care.
Correct positioning of polarity axis in response to internal or external cues is central to cellular morphogenesis and cell fate determination. In the budding yeast Saccharomyces cerevisiae, Bud3p plays a key role in the axial bud-site selection (axial budding) process in which cells assemble the new bud next to the preceding cell division site. Bud3p is thought to act as a component of a spatial landmark. However, it is not clear how Bud3p interacts with other components of the landmark, such as the septins, to control axial budding. Here, we report that overexpression of Bud3p causes the formation of small septin rings (∼1 µm in diameter) and arcs aside from previously reported spiral-like septin structures. Bud3p closely associates with the septins in vivo as Bud3p colocalizes with these aberrant septin structures and forms a complex with two septins, Cdc10p and Cdc11p. The interaction of Bud3p with the septins may involve multiple regions of Bud3p including 1–858, 850–1220, and 1221–1636 a.a. since they all target to the bud neck but exhibit different effects on septin organization when overexpressed. In addition, our study reveals that the axial budding function of Bud3p is mediated by the N-terminal region 1–858. This region shares an amphipathic helix (850–858) crucial for bud neck targeting with the middle portion 850–1103 involved in the formation of ectopic septin spirals and rings. Interestingly, the Dbl-homology domain located in 1–858 is dispensable for axial bud-site selection. Our findings suggest that multiple regions of Bud3p ensure efficient targeting of Bud3p to the bud neck in the assembly of the axial landmark and distinct domains of Bud3p are involved in axial bud-site selection and other cellular processes.
Dendritic cells (DC) are potent professional antigen-presenting cells that drive primary immune responses to infections or other agonists perceived as ‘dangerous’. Muc1 is the only cell surface mucin or MUC gene product that is expressed in DC. Unlike other members of this glycoprotein family, Muc1 possesses a unique cytosolic region capable of signal transduction and attenuating toll-like receptor (TLR) activation. The expression and function of Muc1 has been intensively investigated on epithelial and tumor cells, but relatively little is known about its function on DC. We hypothesized that Muc1 would influence in vitro generation and primary DC activation in response to the TLR4 and TLR5 ligands lipopolysaccharide and flagellin. Compared with Muc1+/+ DC, we found that Muc1−/− DC were constitutively activated, as determined by higher expression of co-stimulatory molecules (CD40, CD80 and CD86), greater secretion of immunoregulatory cytokines (TNF-α and VEGF), and better stimulation of allogeneic naïve CD4+ T cell proliferation. After activation by either LPS or flagellin and co-culture with allogeneic CD4+ T cells, Muc1−/− DC also induced greater secretion of TNF-α and IFN-γ compared to similarly activated Muc1+/+ DC. Taken together, our results indicate that deletion of Muc1 promotes a heightened functional response of DC in response to TLR4 and TLR5 signaling pathways, and suggests a previously under-appreciated role for Muc1 in regulating innate immune responses of DC.
Inflammation; Dendritic cells; Muc1; Toll-like receptor; Innate immunity; Immunomodulation; Host defence; Cytokines
With a national program initiated recently to reduce transmission of Schistosoma japonicum in the People's Republic of China (P.R. China), there is an urgent need for accessible, quality-assured diagnostics for case detection, surveillance, and program monitoring of chemotherapy efficacy and other control interventions in areas of low endemicity. We compared the performance of nine immunodiagnostic tests developed in P.R. China for detection of antibodies against S. japonicum and established their priority for further assessment in field settings.
Using the Kato-Katz technique as the reference standard, 240 well-characterized archived serum specimens (100 positive and 140 negative) were evaluated in nine immunological tests developed in P.R. China. The enzyme-linked immunoelectrotransfer blot assay (EITB), which uses an adult worm extract of S. japonicum, supplied by the Center of Disease Control and Prevention, USA, was also evaluated. The sensitivity and specificity of each test were determined and the reproducibility of each test was assessed by evaluating operator-to-operator and run-to-run variation. In addition the simplicity of use for the end-user was evaluated. All tests showed good sensitivities ranging from 92.0% (95% confidence interval (CI): 86.7–97.3%) to 98.0% (95% CI: 95.3–100.0%). The test specificities varied from 70.0% (95% CI: 62.4–77.6%) to 97.1% (95% CI: 94.4–99.9%). All tests showed excellent reproducibility with a discordant rate in the range of 0–10.0% for operator-to-operator variation and run-to-run variation. All tests, except one magnetic particle-based enzyme-linked immunosorbent assay, were found to be easy to use, especially the dot immunogold filtration assays.
Most evaluated tests had acceptable performance characteristics and could make an impact on the schistosomiasis control programs in P.R. China. Three tests with the highest sensitivity, specificity and greatest ease of use, were selected for further evaluation in field settings.
With the advantages of higher sensitivity and simpler ease of use over stool examination, antibody-detection methods have been integrated into programs for schistosomiasis control in the People's Republic of China after the notable decrease of prevalence and intensity of Schistosoma japonicum infection. We compared the performance of nine immunoassays for diagnosis of S. japonicum using well-characterized archived serum specimens and prioritized tests for future evaluation. Most tests had acceptable performance characteristics and could have an impact on the control of schistosomiasis. Three tests, including one indirect hemagglutination assay, one enzyme-linked immunosorbent assay and one dot immunogold filtration assay were selected for further assessment in field settings. Our final goal is to have appropriate tools for different stages of schistosomiasis control, such as screening targets for chemotherapy, evaluating the efficacy of schistosomiasis control programs, and monitoring the endemic status of schistosomiasis.
Secondary lymphoid organs and peripheral tissues are characterized by hypoxic microenvironments, both in the steady state and during inflammation. Although hypoxia regulates T-cell metabolism and survival, very little is known about whether or how hypoxia influences T-cell activation. We stimulated mouse CD4+ T cells in vitro with antibodies directed against the T-cell receptor (CD3) and CD28 under normoxic (20% O2) and hypoxic (1% O2) conditions. Here we report that stimulation under hypoxic conditions augments the secretion of effector CD4+ T-cell cytokines, especially IFN-γ. The enhancing effects of hypoxia on IFN-γ secretion were independent of mouse strain, and were also unaffected using CD4+ T cells from mice lacking one copy of the gene encoding hypoxia-inducible factor-1α. Using T cells from IFN-γ receptor–deficient mice and promoter reporter studies in transiently transfected Jurkat T cells, we found that the enhancing effects of hypoxia on IFN-γ expression were not due to effects on IFN-γ consumption or proximal promoter activity. In contrast, deletion of the transcription factor, nuclear erythroid 2 p45–related factor 2 attenuated the enhancing effect of hypoxia on IFN-γ secretion and other cytokines. We conclude that hypoxia is a previously underappreciated modulator of effector cytokine secretion in CD4+ T cells.
hypoxia; gene regulation; CD4+ T cells; effector cytokine; IFN-γ
Human major histocompatibility complex class I-related chain A (MICA) plays a dual role in adaptive and innate immune responses. Increasing evidence demonstrates that MICA is closely correlated with acute and chronic kidney allograft rejection. Therefore, understanding the activation mechanisms of MICA is important in kidney transplantation. We previously demonstrated that ischemia/reperfusion injury (IRI) could up-regulate MICA expression on mouse kidney allografts. Since hypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular adaptive responses to hypoxia during IRI, here we investigate whether HIF-1 could up-regulate MICA expression and its influence on NK cell cytotoxicity.
We find that HIF-1alpha plays an important role in up-regulating MICA expression, inducing IFNgamma secretion and NK cell cytotoxicity during hypoxia/reoxygenation. First, we generated a HIF-1alphaDELTAODD-expressing adenovirus to stably and functionally express HIF-1alpha in human renal proximal tubular epithelial (HK-2) cells under normoxia conditions. HIF-1alpha over-expression in HK-2 cells induces MICA expression and enhances NK cell cytotoxic activity towards cells that express HIF-1alpha. Second, we used a hypoxia/reoxygenation cell model to simulate IRI in vitro and found that the suppression of HIF-1alpha by RNAi induces down-regulation of MICA expression and inhibits NK cytotoxicity. In antibody blocking experiments, an anti-MICA mAb was able to down-regulate NK cell cytotoxic activity towards HK-2 cells that over-expressed HIF-1alpha. Moreover, when NK cells were co-cultured with the HK-2 cells expressing MICA, which was up-regulated by over-expression of HIF-1alpha, there was a significant increase in the secretion of IFNgamma. In the presence of the blocking MICA mAb, IFNgamma secretion was significantly decreased.
These results demonstrate that hypoxia/reoxygenation-promoted MICA expression on HK-2 cells is through a HIF-1 pathway. The increased IFNgamma secretion and enhanced NK cell cytotoxicity was mainly due to the surface expression of MICA induced by over-expression of HIF-1alpha. This study enhances our understanding of MICA activation mechanisms during kidney transplantation and provides insights into how IRI can influence transplant outcome. Moreover, these findings might be also important for developing strategies to reduce the effect of MICA in kidney transplant outcomes in the future.